Latent reactive blood compatible agents

ABSTRACT

A reagent and related method for use in passivating a biomaterial surface, the reagent including a latent reactive group and a bifunctional aliphatic acid (e.g., fatty acid), in combination with a spacer group linking the latent reactive group to the aliphatic acid in a manner that preserves the desired function of each group. Once bound to the surface, via the latent reactive group, the reagent presents the aliphatic acid to the physiological environment, in vivo, in a manner (e.g., concentration and orientation) sufficient to hold and orient albumin.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationfiled Apr. 24, 2003 and assigned Ser. No. 10/422,160, which is adivisional of U.S. patent application filed Jul. 29, 2002 and assignedSer. No. 10/207,944, which is a divisional of U.S. patent applicationfiled Oct. 22, 1998 and assigned Ser. No. 09/177,318, which claims thebenefit of provisional U.S. patent application filed Mar. 18, 1998 andassigned Ser. No. 60/078,383, the entire disclosure of which isincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to reagents and methods for rendering asurface biocompatible, and in particular to reagents and methods for“passivating” the surface of an implantable medical device in order torender it hemocompatible. In another aspect, the invention relates tobiomedical devices, per se, and in particular those havingbiocompatible, including hemocompatible, tissue-contacting surfaces.

BACKGROUND OF THE INVENTION

Manufacturers of implantable medical devices have long attempted tounderstand, and in turn improve, the performance of materials used inblood-contacting applications (Leonard, E. F., et al. Ann. N.Y. Acad.Sci. 516, New York, Acad. Sci., New York, 1987). The biological responseof the body, as well as problems with infection, have hindered theapplication of implantable, disposable, and extracorporeal devices.Anticoagulant drugs, such as heparin and coumadin, can improve the useof such devices, although anticoagulants have their own correspondingrisks and drawbacks. For these reasons, development of materials havinggreater compatibility with blood has been pursued aggressively(Sevastianov, V. I., CRC Crit. Rev. Biocomp. 4:109, 1988).

Two general strategies that have been used to develop improvedblood-contacting materials include modifying the chemistry of the bulkmaterial itself, and/or modifying the interfacial properties of thematerial. With regard to the latter approach, several classes ofmaterials have been covalently bonded onto blood-contacting surfaceswith the goal of improving blood compatibility. These includeanticoagulants, such as heparin and hirudin; hydrogels; polyethyleneoxide (PEO); albumin binding agents; cell membrane components;prostaglandins; and sulfonated polymers. These approaches have met withvarying degrees of success in terms of reducing protein adsorption,platelet adhesion and activation, and thrombus formation. Unfortunately,no approach has yet been shown to be universally applicable forimproving blood-biomaterial interactions.

As mentioned above, albumin binding agents have been considered for useon biomaterials. Biomaterials having a high surface concentration ofalbumin have been shown to be less likely to initiate the fibrin cascadeand platelet attachment than those having a high concentration of otherserum proteins, such as fibrinogen, fibronectin, or immunoglobulins. Onmany polymeric materials, however, fibrinogen is often the predominantprotein adsorbed from protein mixtures or plasma. For these reasons,investigators have attempted to immobilize albumin onto materials or todesign biomaterial surfaces that will enhance binding of endogenousalbumin from blood, thus mitigating the adsorption of fibrinogen andconsequent thrombogenic phenomena.

In this respect, a number of different approaches have been employed todate. These approaches include passive adsorption or covalentimmobilization of albumin to the surface, and the development ofsurfaces designed to selectively bind endogenous albumin fromcirculating blood, the latter using alkyl chain-modified materials andother means.

Munro, et al., U.S. Pat. No. 4,530,974, discloses a method of adsorbingalbumin to a water-insoluble polymer such as polyurethane by covalentlybinding to the surface a nonionic hydrophobic aliphatic chain to whichserum albumin will selectively bind.

Frautschi et al., U.S. Pat. No. 5,017,670 and U.S. Pat. No. 5,098,977,teach methods for covalent attachment of aliphatic extensions of 12 to22 carbon atoms to water-insoluble polymers containing aromatic ringsand ring structures with adjacent secondary hydroxyls for increasedalbumin binding.

Eaton, U.S. Pat. No. 5,073,171, describes a biocompatible prostheticdevice incorporating an amount of an albumin binding dye effective toform a coating of endogeneous albumin on the device when the device isin contact with a physiological fluid containing albumin.

While some or all of these various strategies can be used to enhance thebinding of endogenous albumin to blood-contacting material surfaces, andin turn to reduce fibrinogen binding, these approaches are each limitedin one or more respects. Alkyl chain-modified surfaces have been shownto increase albumin binding and decrease fibrinogen binding, but theseeffects were fairly limited, for instance, to a short term time frame(generally less than one hour). In addition, various other surfacemodification methods discussed above are useful for only a narrow rangeof substrate materials.

On another subject, the assignee of this application has developed theability to attach bioactive groups to a surface by covalently bondingthose groups, directly or indirectly, to the surface. For instance, U.S.Pat. Nos. 4,722,906, 4,979,959, 4,973,493 and 5,263,992 relate todevices having biocompatible agents covalently bound via photoreactivegroups and a chemical linking moiety to the biomaterial surface. U.S.Pat. Nos. 5,258,041 and 5,217,492 relate to the attachment ofbiomolecules to a surface through the use of long chain chemicalspacers. U.S. Pat. Nos. 5,002,582 and 5,512,329 relate to thepreparation and use of polymeric surfaces, wherein polymeric agentsproviding desirable properties are covalently bound via a photoreactivemoiety to the surface. In particular, the polymers themselves exhibitthe desired characteristics, and in the preferred embodiment, aresubstantially free of other (e.g., bioactive) groups.

It would be highly desirable to be able to attach albumin to abiomaterial surface in a manner that is suitably stable for extendeduse, particularly in a manner that permits the albumin to be replenishedover time and in the course of use.

SUMMARY OF THE INVENTION

The present invention provides a novel reagent for use in passivating abiomaterial surface, the reagent comprising a latent reactive group anda bifunctional aliphatic acid, in combination with a spacer grouplinking the latent reactive group to the aliphatic acid in a manner thatpreserves the desired function of each group. The reagent can be used topassivate a surface by activating the latent reactive group in thepresence of the surface in order to covalently bond the reagent to thesurface. Once bound to the surface, the reagent presents the aliphaticacid to the physiological environment, in vivo, in a manner (e.g.,concentration and orientation) sufficient to hold and orient albumin.Preferably, over time, the reagent surface is able to replenish itselfby replacing albumin molecules that have become unbound or deterioratedwith new albumin molecules. Albumin (e.g., human serum albumin (HSA)),is defined as any naturally occurring proteinaceous moiety containing afatty acid binding site.

In a preferred embodiment, the reagent is of the general formula(X)_(m)—Y-(Z)_(n) where X is a latent reactive (e.g., photoreactive)group, Y is a spacer radical, and Z is a bifunctional aliphatic acid, aseach are described herein. The values of m and n are ≧1 and while m canequal n, it is not necessary. The aliphatic acid is ‘bifunctional’ inthat it provides both an aliphatic region and an anionic (e.g.,carboxylic acid) region. Once attached to a surface, these portionscooperate in the process of attracting and binding of albumin in orderto passivate the surface.

In the preferred embodiment where both m and n=1, the reagent is termeda heterobifunctional reagent. The aliphatic acid is preferably attachedto the latent reactive group by means of a divalent spacer group in amanner that does not detrimentally affect the function of either thealiphatic or anionic portions. Higher-valent spacer groups can also beselected which permit the attachment of multiple aliphatic acid andlatent reactive groups, again in a manner which does not detrimentallyaffect the functions of the respective groups. In this case m does notnecessarily equal n and both are ≧1.

In a further embodiment, the spacer group can be a multivalent polymerhaving multiple sites along the backbone which permit covalentattachment of the aliphatic acid and latent reactive groups. Thesegroups can be attached to a preformed reactive polymer usingconventional chemical coupling techniques or may be incorporated duringthe polymerization process by use of appropriately substituted monomers.In this embodiment, m does not necessarily equal n and typically bothare larger than one.

The invention further provides a method for preparing a passivatingreagent, as well as a method of using the reagent to passivate thesurface of a synthetic or natural biomaterial. In yet a furtherembodiment, the invention provides a surface coated with a passivatingreagent of this invention, and in turn, an article fabricated from amaterial providing a surface coated or coatable with such a reagent. Inyet a further embodiment, the invention provides a passivatedbiomaterial surface having reagent attached thereto and albuminattracted and attached to the bound reagent.

DETAILED DESCRIPTION

The present invention permits the binding of albumin to a surface to beenhanced by the use of a surface modification reagent. The reagentincludes a bifunctional aliphatic acid capable of being attached to asurface in an amount and orientation that improves the ability of thesurface to attract and bind albumin. While not intending to be bound bytheory, it appears that a surface bearing a reagent of this inventionexhibits improved albumin binding by virtue of both hydrophobicinteractions (of the alkyl chain) and ionic interactions (of the anionicmoiety) with albumin. It is expected that the hydrophobic interactionsserve to hold and orient the free albumin molecule, while the ionicinteractions serve to maintain the albumin molecule in position by theaddition of attractive ionic forces. In a particularly preferredembodiment, the bifunctional aliphatic acid is attached to eitheralkane, oxyalkane, or hydrophobic polymeric backbones to allow bothaliphatic and ionic regions of the bifunctional acid analog to spaciallyorient away from the biomaterial surface to induce better binding withnative albumin. The reagent, in turn, permits albumin binding surfacesto be created using a variety of medical device materials, and inparticular, for use in blood-contacting medical devices.

Bifunctional Aliphatic Acid

The bifunctional aliphatic acid of the present invention (“Z” group)includes both an aliphatic portion and an anionic portion. The word“aliphatic”, as used herein, refers to a substantially linear portion,e.g., a hydrocarbon backbone, capable of forming hydrophobicinteractions with albumin. The word “anionic”, in turn, refers to acharged portion capable of forming further ionic interactions with thealbumin molecule. By the use of a reagent of this invention, theseportions can be covalently attached to a surface in a manner thatretains their desired function, in order to attract and bind nativealbumin from blood and other bodily fluids.

In a preferred embodiment, the invention includes photoactivatiblemolecules having fatty acid functional groups, including polymers havingmultiple photoactivatible and fatty acid functional groups, as well asheterobifunctional molecules. Photoactivatible polyacrylamide copolymerscontaining multiple pendant fatty acid analogs and multiple pendantphotogroups have been synthesized from acrylamide, abenzophenone-substituted acrylamide, and N-substituted acrylamidemonomers containing the fatty acid analog. Photoactivatiblepolyvinylpyrrolidones have also been prepared in a similar fashion.Polyacrylamide or polyvinylpyrrolidone copolymers with a singleend-point photogroup and multiple pendant fatty acid analogs have alsobeen synthesized. Finally, photoactivatible, heterobifunctionalmolecules having a benzophenone on one end and a fatty acid group on theother end optionally separated by a spacer have been made, wherein thatspacer can be a hydrophobic alkyl chain or a more hydrophilicpolyethyleneglycol (PEG) chain.

Spacer Group

Suitable spacers (“Y” groups) for use in preparing heterobifunctionalreagents of the present invention include any di- or higher-functionalspacers capable of covalently attaching a latent reactive group to analiphatic acid in a manner that permits them both to be used for theirintended purpose. Although the spacer may itself provide a desiredchemical and/or physical function, preferably the spacer isnon-interfering, in that it does not detrimentally affect the use of thealiphatic and ionic portions for their intended purposes. In the case ofthe polymeric reagents of the invention, the spacer group serves toattach the aliphatic acid to the backbone of the polymer.

The spacer may be either aliphatic or polymeric and contain variousheteroatoms such as O, N, and S in place of carbon. Constituent atoms ofthe spacers need not be aligned linearly. For example, aromatic rings,which lack abstractable hydrogen atoms (as defined below), can beincluded as part of the spacer design in those reagents where the latentreactive group functions by initiating covalent bond formation viahydrogen atom abstraction. In its precursor form (i.e., prior toattachment of a photoreactive group and aliphatic acid), a spacer can beterminated with any suitable functionalities, such as hydroxyl, amino,carboxyl, and sulfhydryl groups, which are suitable for use in attachinga photoreactive group and the aliphatic acid by a suitable chemicalreaction, e.g., conventional coupling chemistry.

Alternatively, the spacer can be formed in the course of combining aprecursor containing (or capable of attaching) the photoreactive groupwith another containing (or capable of attaching) the aliphatic acid.For example, the aliphatic acid could be reacted with an aliphaticdiamine to give an aliphatic amine derivative of the bifunctionalaliphatic acid and which could be coupled with a carboxylic acidcontaining the photogroup. To those skilled in the art, it would beobvious that the photogroup could be attached to any appropriatethermochemical group which would react with any appropriate nucleophilecontaining O, N or S.

Examples of suitable spacer groups include, but are not limited to, thegroups consisting of substituted or unsubstituted alkylene, oxyalkylene,cycloalkylene, arylene, oxyarylene, or aralkylene group, and havingamides, ethers, and carbonates as linking functional groups to thephotoactivatible group, and the bifunctional aliphatic fatty acid.

The spacer of the invention can also comprise a polymer which serves asa backbone. The polymer backbone can be either synthetic or naturallyoccurring, and is preferably a synthetic polymer selected from the groupconsisting of oligomers, homopolymers, and copolymers resulting fromaddition or condensation polymerization. Naturally occurring polymers,such as polysaccharides, can be used as well. Preferred backbones arebiologically inert, in that they do not provide a biological functionthat is inconsistent with, or detrimental to, their use in the mannerdescribed.

Such polymer backbones can include acrylics such as those polymerizedfrom hydroxyethyl acrylate, hydroxyethyl methacrylate, glycerylacrylate, glyceryl methacrylate, acrylic acid, methacrylic acid,acrylamide and methacrylamide; vinyls such as polyvinylpyrrolidone andpolyvinyl alcohol; nylons such as polycaprolactam; derivatives ofpolylauryl lactam, polyhexamethylene adipamide and polyhexamethylenedodecanediamide, and polyurethanes; polyethers such as polyethyleneoxide, polypropylene oxide, and polybutylene oxide; and biodegradablepolymers such as polylactic acid, polyglycolic acid, polydioxanone,polyanhydrides, and polyolthoesters.

The polymeric backbone is chosen to provide a backbone capable ofbearing one or more photoreactive groups, and one or more fatty acidfunctional groups. The polymeric backbone is also selected to provide aspacer between the surface and the various photoreactive groups andfatty acid functional groups. In this manner, the reagent can be bondedto a surface or to an adjacent reagent molecule, to provide the fattyacid functional groups with sufficient freedom of movement todemonstrate optimal activity. The polymer backbones are preferably watersoluble, with polyacrylamide and polyvinylpyrrolidone being particularlypreferred polymers.

Photoreactive Group

In a preferred embodiment one or more photoreactive groups are providedby the X groups attached to the central Y spacer radical. Upon exposureto a suitable light source, each of the photoreactive groups are subjectto activation. The term “photoreactive group”, as used herein, refers toa chemical group that responds to an applied external energy source inorder to undergo active specie generation, resulting in covalent bondingto an adjacent chemical structure (e.g., an aliphatic carbon-hydrogenbond).

Preferred X groups are sufficiently stable to be stored under conditionsin which they retain such properties. See, e.g., U.S. Pat. No.5,002,582, the disclosure of which is incorporated herein by reference.Latent reactive groups can be chosen that are responsive to variousportions of the electromagnetic spectrum, with those responsive toultraviolet and visible portions of the spectrum (referred to herein as“photoreactive”) being particularly preferred.

Photoreactive aryl ketones are preferred, such as acetophenone,benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles(i.e., heterocyclic analogues of anthrone such as those having N, O, orS in the 10-position), or their substituted (e.g., ring substituted)derivatives. The functional groups of such ketones are preferred sincethey are readily capable of undergoing theactivation/inactivation/reactivation cycle described herein.Benzophenone is a particularly preferred photoreactive group, since itis capable of photochemical excitation with the initial formation of anexcited singlet state that undergoes intersystem crossing to the tripletstate. The excited triplet state can insert into carbon-hydrogen bondsby abstraction of a hydrogen atom (for example, from a support surfaceor target molecule in the solution and in bonding proximity to theagent), thus creating a radical pair. Subsequent collapse of the radicalpair leads to formation of a new carbon-carbon bond. If a reactive bond(e.g., carbon-hydrogen) is not available for bonding, the ultravioletlight-induced excitation of the benzophenone group is reversible and themolecule returns to ground state energy level upon removal of the energysource. Hence, photoreactive aryl ketones are particularly preferred.

The azides constitute a preferred class of latent reactive groups andinclude arylazides (C₆R₅N₃) such as phenyl azide and particularly4-fluoro-3-nitrophenyl azide, acyl azides (—CO—N₃) such as ethylazidoforinate, phenyl azidoformate, sulfonyl azides (—SO₂—N₃) such asbenzenesulfonyl azide, and phosphoryl azides (RO)₂PON₃ such as diphenylphosphoryl azide and diethyl phosphoryl azide. Diazo compoundsconstitute another class of photoreactive groups and includediazoalkanes (—CHN₂) such as diazomethane and diphenyldiazomethane,diazoketones (—CO—CHN₂) such as diazoacetophenone and1-trifluoromethyl-1-diazo-2-pentanone, diazoacetates (—CO—CN₂—CO—O—)such as t-butyl alpha diazoacetoacetate. Other photoreactive groupsinclude aliphatic azo compounds such as azobisisobutyronitrile,diazirines (—CHN₂) such as 3-trifluoromethyl-3-phenyldiazirine andketenes (—CH═C═O) such as ketene and diphenylketene.

Upon activation of the photoreactive groups, the coating adhesionmolecules are covalently bound to each other and/or to the materialsurface by covalent bonds through residues of the photoreactive groups.Exemplary photoreactive groups, and their residues upon activation, areshown as follows. Photoreactive Group Residue Functionality aryl azidesamine R—NH—R′ acyl azides amide R—CO—NH—R′ azidoformates carbamateR—O—CO—NH—R′ sulfonyl azides sulfonamide R—SO₂—NH—R′ phosphoryl azidesphosphoramide (RO)₂PO—NH—R′ diazoalkanes new C—C bond diazoketones newC—C bond and ketone diazoacetates new C—C bond and esterbeta-keto-alpha-diazoacetates new C—C bond and beta-ketoester aliphaticazo new C—C bond diazirines new C—C bond ketenes new C—C bondphotoactivated ketones new C—C bond and alcoholPreparation of Reagents

Reagents of the present invention can be prepared by any suitable means,depending upon the selection of either a heterobifunctional reagent or apolymeric reagent. In the case of the heterobifunctional reagents, thefatty acid residue is provided by a fatty acid possessing a chemicallyreactive group on the alkyl chain which permits covalent coupling of theremainder of the heterobifunctional molecule to the fatty acid withpreservation of the carboxylic acid functionality. Preferably, the siteof the reactive group is in close proximity to the carboxylic acid groupso as to minimize effects on the binding activity of the hydrophobicalkyl chain. Most preferably, the fatty acid residue can be provided bya compound such n-tetradecylsuccinic anhydride (TDSA). Reaction of sucha molecule with a second molecule possessing a nucleophilic species suchas a primary amine results in opening of the anhydride ring to give afatty acid with an amide linkage to the remainder of the molecule. Thisreaction generates a pair of regioisomers depending upon the directionof the anhydride ring opening. The second molecule in this reaction canbe provided by a spacer group, with or without a photoactivatible group,which possesses a group capable of reaction with the fatty acidcompound. Most preferably, this spacer group possesses an amine which ishighly reactive with an anhydride species. The spacer group is typicallya bifunctional molecule which can have the photoactivatible groupattached prior to reaction with the fatty acid derivative or the reverseorder of reaction can be used. The bifunctional spacer can be eitherheterobifunctional or homobifunctional, with the former requiring adifferential reactivity in the first and second reaction steps and thelatter requiring an efficient method of separating themonofunctionalized spacer following the first reaction. Optionally, nospacer is required and a photoactivatible group possessing functionalitycapable of reaction with the fatty acid derivative can be used. Theabove examples are nonlimiting and the methods of accomplishing thesecoupling reactions are apparent to those skilled in the art.

Polymeric reagents of the invention can be prepared by derivatization ofpreformed polymers possessing reactive groups along the backbone of thepolymer capable of reaction with the photoactivatible groups and thefatty acid derivatives. For example, polyacrylamide,polyvinylpyrrolidone, or siloxanes functionalized with amine groupsalong the backbone, with or without a spacer group, can be reacted with4-benzoylbenzoyl chloride (BBA-Cl) and TDSA to provide thephotoactivatible and fatty acid ligands respectively. Alternatively, thephotoactivatible and fatty acid groups can be prepared in the form ofpolymerizable monomers which can then be copolymerized with themselvesand other monomers to provide polymers of the invention. In a furtherembodiment of the invention, the photoactivatible group can beintroduced in the form of a chain transfer agent along with the fattyacid monomer and other comonomers so as to provide a polymer having thephotoactivatible group at the end of the polymer chain. For example, achain transfer agent possessing two derivatized benzophenones as thephotoactivatible groups and a mercaptan as the chain transfer agent canbe used to copolymerize a fatty acid monomer and acrylamide orN-vinylpyrrolidone monomers to provide polymers of the invention.Alternatively, this polymer could be prepared with reactive groups alongthe backbone, followed by reaction with a fatty acid derivative.

Surfaces and Methods of Attachment.

The reagent of the present invention can be used to modify any suitablesurface. Where the latent reactive group is a photoreactive group of thepreferred type, it is particularly preferred that the surface provideabstractable hydrogen atoms suitable for covalent bonding with theactivated group.

Plastics such as polyolefins, polystyrenes, poly(methyl)methacrylates,polyacrylonitriles, poly(vinylacetates), poly (vinyl alcohols),chlorine-containing polymers such as poly(vinyl) chloride,polyoxymethylenes, polycarbonates, polyamides, polyimides,polyurethanes, phenolics, amino-epoxy resins, polyesters, silicones,cellulose-based plastics, and rubber-like plastics can all be used assupports, providing surfaces that can be modified as described herein.See generally, “Plastics”, pp. 462-464, in Concise Encyclopedia ofPolymer Science and Engineering, Kroschwitz, ed., John Wiley and Sons,1990, the disclosure of which is incorporated herein by reference. Inaddition, supports such as those formed of pyrolytic carbon andsilylated surfaces of glass, ceramic, or metal are suitable for surfacemodification.

Any suitable technique can be used for reagent binding to a surface, andsuch techniques can be selected and optimized for each material,process, or device. The reagent can be successfully applied to cleanmaterial surfaces as listed above by spray, dip, or brush coating of asolution of the fatty acid binding reagent. The surface may be air-driedprior to illumination or the surface can be illuminated while submergedin the coating solution. The photoreactive group is energized via anexternal stimulation (e.g., exposure to a suitable light source) toform, via free active specie generation, a covalent bond between thereagent and either another polybifunctional reagent molecule or thebiomaterial surface. This coating method is herein termed the “one stepcoating method”, since photoreactive coupling chemistry attaches aninvention polymer to a biomaterial surface, and no subsequent steps arerequired to add the bioactive group. The external stimulation that isemployed desirably is electromagnetic radiation, and preferably isradiation in the ultraviolet, visible or infrared regions of theelectromagnetic spectrum.

The “two-step” method would involve a first step of photocoupling ahydrocarbon backbone to the surface, followed by a second step ofattaching (e.g., thermochemically) one or more fatty acid derivatives tothe immobilized backbone. For example, this two step approach couldinvolve covalently attaching a photoreactive hydrocarbon backbonecontaining nucleophiles which could be used to thermochemically couplefatty acid derivatives to the surface, or directly attachingthermochemical groups (e.g. amines) to the surface, followed bythermochemical attachment of one or more fatty acid derivatives.

Alternatively, chemically reactive groups can be introduced on thesurface by a variety of non-photochemical methods, followed by chemicalcoupling of the fatty acid group to the modified surface. For example,amine groups can be introduced on a surface by plasma treatment with amixture of methane and ammonia and the resulting amines can then bereached with TDSA to chemically couple the fatty acid derivative to thesurface through an amide linkage. When desired, other approaches can beused for surface modification using the reagent of the presentinvention. This approach is particularly useful in those situations inwhich a support is difficult to modify using conventional chemistry, orfor situations that require exceptional durability and stability of thetarget molecule on the surface.

EXAMPLES

The invention will be further described with reference to the followingnon-limiting Examples, which incorporate the following table offormulas. It will be apparent to those skilled in the art that manychanges can be made in the embodiments described without departing fromthe scope of the present invention. Thus the scope of the presentinvention should not be limited to the embodiments described in thisapplication, but only by embodiments described by the language of theclaims and the equivalents of those embodiments. Unless otherwiseindicated, all percentages are by weight. Compound/ Formula ExampleNotation

1/1 4-Benzoylbenzoyl chloride

2/2 4-Bromoethyl- benzophenone

3/3 Poly(ethylene glycol)₂₀₀Mono-4- benzoylbenzyl Ether

4/4 Poly(ethylene glycol)₂₀₀Mono-4- benzoylbenzyl EtherMonomethanesulfonate

5/5 Monoaminopoly (ethylene glycol)₂₀₀ Mono-4- benzoylbenzyl Ether

6/6 Mono-2- (carboxymethyl) hexadecanamidopoly (ethyleneglycol)₂₀₀Mono-4- benzoylbenyl Ether

7/6 Mono-3- carboxyhepta- decanamidopoly (ethylene glycol)₂₀₀Mono-4-benzoylbenzyl Ether

8/7 Mono-2- (carboxymethyl) hexadecanamidotetra (ethylene glycol)Mono-4- benzoylbenzyl Ether

9/7 Mono-3- carboxyhepta- decanamidotetra (ethylene glycol) Mono-4-benzoylbenzyl Ether

10/8  N-[2-(4- Benzoylbenzyloxy) ethyl]-2- (carboxymethyl)hexadecanamide

11/8  N-[2-(4- Benzoylbenzyloxy) ethyl]-3- carboxylhepta- decanamide

12/9  N-[12- (Benzoylbenzyloxy) dodecyl]-2- (carboxymethyl)hexadecanamide

13/9  N-[12- (Benzoylbenzyloxy) dodecyl]-3-carboxy- heptadecanamide

14/10 N-[3-(4- Benzoylbenzamido) propyl]-2- (carboxymethyl)hexadecanamide

15/10 N-[3-(4- Benzoylbenzamido) propyl]-3- carboxyhepta- decamide

16/11 N-(3-Benzoylphenyl)- 2-(carboxymethyl) hexadecanamide

17/11 N-(3-Benzoylphenyl)- 3-carboxyhepta- decanamide

18/12 N-(4-Benzoylphenyl)- 2-(carboxymethyl) hexadecanamide

19/12 N-(4-Benzoylphenyl)- 3-carboxyhepta- decanamide

20/13 Monohexadecanamido- poly(ethylene glycol)₂₀₀Mono-4- benzoylbenzylEther

21/14 Mono-3- Carboxypropana- midpoly (ethylene glycol)₂₀₀Mono-4-benzoylbenzyl Ether

22/15 Hexadecyl 4- benzoylbenzil ether

23/16 Poly(ethylene glycol)₂₀₀Monohexadecyl Mono-4- benzoylbenzyl Ether

24/17 Poly(ethylene glycol)₂₀₀Mono-15- carboxypentadecyl Mono-4-benzoylbenzyl Ether

25/18 Mono-15- carboxypenta- decanamidopoly (ethylene glycol)₂₀₀ Mono-4-benzoylbenzyl Ether

26/19 N-[3- Methacrylamido) propyl]-2- (carboxymethyl)hexade- canamide

27/19 N-[3- Methacrylamido) propyl]-3- carboxyhepta- decanamide

28/20 N-[3-(4- Benzoylbenzamido) propyl]methacrylamide

29/21 N-(2-Mercaptoethyl)- 3,5-bis(4- benzoylbenzyloxy) benzamide

30/22 Photoreactive Endpoint Copolymer of Acrylamide and Fatty AcidMonomers

31/23 Photoreactive Random Copolymer of Acrylamide and Fatty AcidMonomers

32A-C/24 Photoreactive Endpoint Copolymer of N-Vinylpyrrolidone andFatty Acid Monomers

33A-D/25 Photoreactive Random Copolymer of N-Vinylpyrrolidone and FattyAcid Monomers

34/26 Photoreactive Siloxane Copolymer Containing Fatty Acids Ligands

Example 1 Preparation of 4-Benzoylbenzoyl Chloride (BBA-Cl) (Compound 1)

4-Benzoylbenzoic acid (BBA), 1.0 kg (4.42 moles), was added to a dry 5liter Morton flask equipped with reflux condenser and overhead stirrer,followed by the addition of 645 ml (8.84 moles) of thionyl chloride and725 ml of toluene. Dimethylformamide (DMF), 3.5 ml, was then added andthe mixture was heated at reflux for 4 hours. After cooling, thesolvents were removed under reduced pressure and the residual thionylchloride was removed by three evaporations using 3×500 ml of toluene.The product was recrystallized from toluene/hexane (1/4) to give 988 g(91% yield) after drying in a vacuum oven. Product melting point was92-94° C. Nuclear magnetic resonance (NMR) analysis at 80 MHz wasconsistent with the desired product. The final compound was stored foruse in the preparation of photoactivatable compounds, as described forinstance in Examples 10 and 20.

Example 2 Preparation of 4-Bromomethylbenzophenone (BMBP) (Compound 2)

4-Methylbenzophenone, 750 g (3.82 moles), was added to a 5 liter Mortonflask equipped with an overhead stirrer and dissolved in 2850 ml ofbenzene. The solution was then heated to reflux, followed by thedropwise addition of 610 g (3.82 moles) of bromine in 330 ml of benzene.The addition rate was approximately 1.5 ml/min and the flask wasilluminated with a 90 watt (90 joule/sec) halogen spotlight to initiatethe reaction. A timer was used with the lamp to provide a 10% duty cycle(on 5 seconds, off 40 seconds), followed in one hour by a 20% duty cycle(on 10 seconds, off 40 seconds). At the end of the addition, the productwas analyzed by gas chromatography and was found to contain 71% of thedesired 4-bromomethylbenzophenone, 8% of the dibromo product, and 20%unreacted 4-methylbenzophenone. After cooling, the reaction mixture waswashed with 10 g of sodium bisulfite in 100 ml of water, followed bywashing with 3×200 ml of water. The product was dried over sodiumsulfate and recrystallized twice from toluene/hexane (1/3 by volume(v/v)). After drying under vacuum, 635 g of BMBP were isolated,providing a yield of 60% and having a melting point of 112-114° C.Analysis on an NMR spectrometer was consistent with the desired product.The final compound was stored for use in the preparation ofphotoactivatable compounds, as described for instance in Examples 3, 7,8, 9, 15, and 21.

Example 3 Preparation of Poly(ethylene glycol)₂₀₀ Mono-4-benzoylbenzylEther (Compound 3)

The poly(ethylene glycol)₂₀₀ (PEG), 72.72 g (0.363 mol), was azeotropedwith 200 ml of toluene for two hours to remove moisture, followed by theremoval of excess toluene under vacuum. The PEG residue was thendissolved in 400 ml of anhydrous tetrahydrofuran (THF) while stirringunder argon at 4° C. Sodium hydride, 2.90 g of a 60% mixture in mineraloil (72.5 mmol), was added in portions and the mixture was stirred 1hour at room temperature. BMBP, 20.0 g (72.7 mmol), prepared accordingto the general method described in Example 2, was added as a solution in100 ml of THF over a 2 hour period and the mixture was stirred 16 hoursat room temperature under argon. The reaction was quenched with aqueousammonium chloride (36 g in 200 ml of water) and the organic solvent wasremoved under vacuum. The residue was dissolved in brine, extracted withchloroform, and the resulting organic extracts were dried over sodiumsulfate. The product was isolated as a viscous oil by adding thechloroform solution to diethyl ether, resulting in precipitation of27.64 g of the desired product. The product was used without additionalpurification. Analysis on an NMR spectrometer was consistent with thedesired product.

Example 4 Preparation of Poly(ethylene glycol₂₀₀ Mono-4-benzoylbenzylEther Monomethanesulfonate (Compound 4)

Compound 3, 3.0 g (7.61 mmol), prepared according to the general methoddescribed in Example 3, was dissolved in 25 ml of methylene chloride,followed by the addition of 1.5 g (14.8 mmol) of triethylamine (TEA).The mixture was cooled on an ice bath under argon and 1.3 g (11.3 mmol)of methanesulfonyl chloride (MsCl) were added dropwise over a 10 minuteperiod. The reaction temperature was allowed to rise to ambienttemperature overnight. The precipitated salts were removed by filtrationand the solvent was removed under vacuum. The residue was dissolved intoluene and filtered to remove solids, followed by evaporation undervacuum to give 3.01 g of product. No further purification of the productwas done at this point. Analysis on an NMR spectrometer was consistentwith the desired product.

Example 5 Preparation of Monoaminopoly(ethylene glycol)₂₀₀Mono-4-benzoylbenzyl Ether (Compound 5)

Compound 4, 17.97 g (38.07 mmol), prepared according to the generalmethod described in Example 4, was dissolved in 100 ml of anhydrous THFin a thick-walled tube, followed by the addition of 100 ml ofconcentrated ammonium hydroxide. The tube was sealed and the two phasemixture was stirred vigorously at 65° C. for 16 hours. The solvent wasremoved under vacuum and the resulting residue was dissolved inchloroform. The product was loaded on a silica gel flash chromatographycolumn and eluted with chloroform/acetone/acetic acid (60/40/1 v/v)until all of the less polar impurities were removed. The product wasthen eluted with chloroform/methanol/acetic acid/water (85/15/1/1 v/v).The fractions which were UV, ninhydrin, and Dragendorff positive werepooled and the solvent was removed under vacuum to give 8.63 g ofproduct. Analysis on an NMR spectrometer was consistent with the desiredproduct.

Example 6 Preparation ofMono-2-(carboxymethyl)hexadecanamidopoly(ethylene glycol)₂₀₀Mono-4-benzoylbenzyl Ether (Compound 6) andMono-3-carboxyheptadecanamidopoly(ethylene glycol₂₀₀Mono-4-benzoylbenzyl Ether (Compound 7)

Compound 5, 3.03 g (7.71 mmol), prepared according to the general methoddescribed in Example 5, and TEA, 2.24 g (22.1 mmol), were dissolved in30 ml of methylene chloride, followed by the addition of 2.40 g (8.10mmol) of TDSA as the solid. The reaction mixture was stirred 18 hours atroom temperature under argon. The solvents were removed under vacuum andthe resulting oil was purified by silica gel flash chromatography usinga solvent gradient: 500 ml of ether/hexane (75/25 v/v); 500 ml ofether/hexane/acetic acid (75/25/1 v/v); chloroform/acetone/acetic acid(60/40/1 v/v); and chloroform/methanol/acetic acid/water (85/15/1/1v/v). The fractions were pooled to give two separate UV and Dragendorffpositive materials representing the regioisomers resulting from ringopening of the anhydride ring. Evaporation of solvent gave 1.35 g ofproduct in one fraction and 0.893 g in the second. Analysis on an NMRspectrometer was consistent with the desired products.

Example 7 Preparation ofMono-2-(carboxymethyl)hexadecanamidotetra(ethyleneglycol)Mono-4-benzoylbenzyl Ether (Compound 8) andMono-3-carboxyheptadecanamidotetra(ethylene glycol) Mono-4-benzoylbenzylEther (Compound 9)

The tetraethylene glycol (TEG), 7.063 g (36.4 mmol), was azeotroped with200 ml of toluene for two hours to remove moisture, followed by theremoval of excess toluene under vacuum. The TEG residue was thendissolved in 70 ml of anhydrous THF while stirring under argon on an icebath. Sodium hydride, 1.45 g of a 60% mixture in mineral oil (36.3mmol), was added and the mixture was stirred 1 hour at room temperature.BMBP, 5.0 g (18.2 mmol), prepared according to the general methoddescribed in Example 2, was added and the mixture was stirred 16 hoursat room temperature under argon. The reaction was quenched with aqueousammonium chloride (9 g in 40 ml of water) and the organic solvent wasremoved under vacuum. The residue was dissolved in saturated brine,extracted with chloroform, and the resulting organic extracts were driedover sodium sulfate. The product was isolated as a viscous oil by addingthe chloroform solution to diethyl ether. The crude product, 7.6 g, wasused without additional purification.

The entire product from above was dissolved in 200 ml of methylenechloride, followed by the addition of 3.96 g (39.1 mmol) of TEA. Themixture was cooled to 4° C. under argon and 3.35 g (29.2 mmol) of MsClwere added. After 6 hours, an additional 1 ml each of TEA and MsCl wereadded and the reaction was left to stir for 16 hours to insure completereaction. The precipitated salts were removed by filtration and thesolvent was removed under vacuum. The residue was dissolved in tolueneand filtered to remove solids, followed by evaporation under vacuum. Nofurther purification of the product was done at this point.

The entire mesylate product from above was dissolved in 50 ml of THF ina thick-walled glass tube, followed by the addition of 50 ml ofconcentrated ammonium hydroxide. The tube was sealed and the two phasemixture was stirred vigorously at 65° C. for 16 hours. The solvent wasremoved under vacuum and the resulting residue was dissolved in 20 ml ofchloroform. After drying over sodium sulfate, the product wasprecipitated by addition of the chloroform solution to diethyl etherresulting in approximately 4.5 g of a brown viscous oil. A portion ofthe product, approximately 1 g, was purified by silica gel flashchromatography using a solvent gradient of ether/hexane/acetic acid(75/25/1 v/v), followed by chloroform/acetone/acetic acid (60/40/1 v/v),and chloroform/ethanol/water/acetic acid (85/15/1/1 v/v). A total of 220mg of purified product were isolated. Analysis on an NMR spectrometerwas consistent with the desired product.

The amine product from above, 0.220 g (0.568 mmol), and TEA, 63 mg(0.623 mmol), were dissolved in 20 ml of methylene chloride, followed bythe addition of 0.185 g (0.625 mmol) of TDSA. The reaction mixture wasstirred 48 hours at room temperature under argon. The solvents wereremoved under vacuum and the resulting oil was purified by silica gelflash chromatography using an chloroform/methanol/water/acetic acid(85/15/1/1 v/v). The appropriate fractions were pooled, evaporated,redissolved in chloroform, and dried over sodium sulfate. Evaporation ofsolvent gave 234 mg of a waxy solid as a mixture of regioisomersresulting from opening of the anhydride ring. Analysis on an NMRspectrometer was consistent with the desired products.

Example 8 Preparation ofN-[2-(4-Benzoylbenzyloxy)ethyl]-2-(carboxymethyl)hexadecanamide(Compound 10) andN-[2-(4-Benzoylbenzyloxy)ethyl]-3-carboxylheptadecanamide (Compound 11)

Anhydrous ethanolamine, 1.00 g (16.4 mmol), was dissolved in 5 ml ofanhydrous THF with stirring under argon. Sodium hydride, 0.655 g (16.4mmol) of a 60% mineral oil dispersion, was added as a solid followed byan additional 5 ml of anhydrous THF. The resulting mixture was stirredat room for 45 minutes at which time no more hydrogen evolution wasobserved. The BMBP, 4.50 g (16.4 mmol), prepared according to thegeneral method described in Example 2, was added as a solution in 25 mlof THF over a 30 minute period. The reaction was allowed to stirovernight at room temperature. The reaction was quenched with water andthe product was extracted with chloroform. The organic extract waswashed with 0.1 N HCl and the aqueous solution was washed one time withchloroform. The aqueous was then evaporated under vacuum, dissolved in10% methanol in chloroform (v/v) and dried over sodium sulfate.Evaporation of solvent gave 2.62 g of a pale yellow solid which was usedwithout additional purification.

The above amine, 0.625 g (2.14 mmol), and TDSA, 0.467 g (1.57 mmol),were dissolved in 10 ml of methylene chloride, followed by the additionof 660 μl (4.74 mmol) of TEA. The resulting solution was stirred at roomtemperature for 16 hours to complete the reaction. The product wasdiluted with water and treated with 5% HCl, followed by separation ofthe organic layer and drying over sodium sulfate. The solvent wasremoved under vacuum and the product was purified using silica gel flashchromatography with a solvent gradient of chloroform followed by 2.5%and 5% (v/v) methanol in chloroform. The appropriate fractions werepooled to give 357 mg of product as a pair of regioisomers resultingfrom the opening of the anhydride ring. Analysis on an NMR spectrometerwas consistent with the desired products.

Example 9 Preparation ofN-[12-(Benzoylbenzyloxy)dodecyl]-2-(carboxymethyl)hexadecanamide(Compound 12) andN-[12-(Benzoylbenzyloxy)dodecyl]-3-carboxyheptadecanamide (Compound 13)

1,12-Dodecanediol, 5.0 g (24.7 mmol), was dissolved in 50 ml ofanhydrous THF in a dry flask under nitrogen. The sodium hydride, 0.494 gof a 60% dispersion in mineral oil (12.4 mmol), was added in portionsover a five minute period. The resulting mixture was stirred at roomtemperature for one hour. BMBP, 3.40 g (12.4 mmol), prepared accordingto the general method described in Example 2, was added as a solid alongwith sodium iodide (0.185 g, 1.23 mmol) and tetra-n-butylammoniumbromide (0.398 g, 1.23 mmol). The mixture was stirred at a gentle refluxfor 24 hours. The reaction was then cooled, quenched with water,acidified with 5% HCl, and extracted with chloroform. The organicextracts were dried over sodium sulfate and the solvent was removedunder vacuum. The product was purified on a silica gel flashchromatography column using chloroform to elute non-polar impurities,followed by elution of the product with chloroform/ethyl acetate (80/20v/v). Pooling of appropriate fractions and evaporation of solvent gave3.42 g of product, a 70% yield. Analysis on an NMR spectrometer wasconsistent with the desired product.

The above alcohol, 1.30 g (3.28 mmol), was dissolved in 13 ml ofanhydrous methylene chloride, followed by 0.829 g (8.19 mmol) of TEA andcooling on an ice bath under argon. MsCl, 0.563 g (4.91 mmol), was addeddropwise over a five minute period, followed by stirring at roomtemperature for 16 hours. The reaction was diluted with water, acidifiedwith 5% HCl, and extracted with chloroform. The organic extracts weredried over sodium sulfate and evaporated to give 1.56 g of a yellow oil.This product was used without further purification. Analysis on an NMRspectrometer was consistent with the desired product.

The above mesylate, 1.56 g (3.28 mmol), was dissolved in 25 ml of THF ina thick-walled tube, followed by the addition of 25 ml of ammoniumhydroxide. The tube was sealed and the mixture was stirred vigorouslyfor 72 hours at 80° C. The mixture was treated with 200 ml of water andthe product was extracted with chloroform. The organic extracts weredried over sodium sulfate and the product was purified on a silica gelflash chromatography column. The column was eluted with chloroform andchloroform/methanol (95/5 v/v) until the less polar impurities wereremoved, followed by elution of the desired product usingchloroform/methanol/ammonium hydroxide (70/25/5 v/v). Pooling of theninhydrin and UV active fractions and evaporation of solvent gave 0.526g of product, a 40% yield. Analysis on an NMR spectrometer wasconsistent with the desired product.

The above amine, 0.440 g (1.11 mmol), was dissolved in 7 ml of methylenechloride, followed by 0.329 g (1.11 mmol) of TDSA and 0.337 g (3.33mmol) of TEA. The resulting mixture was stirred at room temperature for36 hours. The reaction was then diluted with water, acidified with 5%HCl, and extracted with chloroform. The organic extracts were dried oversodium sulfate and the residue after evaporation was purified on silicagel flash chromatography. A solvent gradient of chloroform, 2.5%methanol in chloroform (v/v), and 5% methanol in chloroform (v/v) wasused to elute the product. A total of 378 mg of product were isolated asa partially resolved pair of regioisomers resulting from opening of theanhydride ring. Analysis on an NMR spectrometer was consistent with thedesired products.

Example 10 Preparation ofN-[3-(4-Benzoylbenzamido)propyl]-2-(carboxymethyl)hexadecanamide(Compound 14) andN-[3-(4-Benzoylbenzamido)propyl]-3-carboxyheptadecanamide (Compound 15)

1,3-Diaminopropane, 1.910 kg (25.77 mol), was placed in a 12 literMorton flask and diluted with 1000 ml of methylene chloride. Aftercooling to below 10° C. on an ice bath, a solution of 1.005 kg (5.175mol) of t-butyl phenyl carbonate in 250 ml of methylene chloride wasadded slowly to the diamine while keeping the temperature below 15° C.at all times. Once the addition was complete, the mixture was warmed toroom temperature for 2 hours to complete the reaction. The reaction wasfurther diluted with 900 ml of methylene chloride, followed by theaddition of 500 g of ice and a slow addition of 2500 ml of 2.2 N NaOH.The organic layer was separated and the basic aqueous solution wasextracted with 3×1250 ml of methylene chloride, keeping each extractseparate. Each of these separate extracts was successively washed with1250 ml of 0.6 N NaOH, beginning with the first extract and proceedingto the last. This wash procedure was repeated and the organic extractswere combined and dried over sodium sulfate. Evaporation of solventyielded 825 g of product for a 92% yield. This product was used withoutany further purification. Analysis on an NMR spectrometer was consistentwith the desired product.

The above amine, 0.774 g (4.44 mmol), was diluted with 20 ml ofanhydrous methylene chloride, followed by the addition of 1.24 g (12.3mmol) of TEA and a dropwise addition of 10 ml of anhydrous methylenechloride containing of 1.0 g (4.09 mmol) of BBA-CL, prepared accordingto the general method described in Example 1, After stirring 1.5 hoursat room temperature, the reaction was diluted with water and acidifiedwith 1 N HCl. The product was extracted with chloroform and the organicextracts were dried over sodium sulfate. Silica gel flash chromatographyusing chloroform/methanol (90/10 v/v) gave 1.68 g of product, slightlygreater than theoretical because of solvent residues. Mass spectralanalysis confirmed the desired product.

The above product, 1.5 g (3.95 mmol), was dissolved in 10 ml oftrifluoroacetic acid under a nitrogen atmosphere. After stirring 3 hoursat room temperature to remove the t-butyloxycarbonyl (t-BOC) protectinggroup, the solvent was removed under reduced pressure and the productwas purified using silica gel flash chromatography. After removal of theless polar impurities with chloroform/methanol (90/10 v/v), the elutingsolvent was switched to chloroform/methanol/ammonium hydroxide (70/25/5v/v) for isolation of the desired product. Pooling of the appropriatefractions and evaporation of solvent gave 1.77 g of product. Analysis onan NMR spectrometer was consistent with the desired product.

A portion of above amine product, 0.500 g (1.77 mmol), was dissolved in10 ml of anhydrous methylene chloride under an argon atmosphere. TEA,0.197 g (1.95 mmol), was added, followed by 0.577 g (1.95 mmol) of TDSA.The reaction was stirred for four hours at room temperature. The mixturewas diluted with water, extracted with methylene chloride, and theorganic extracts were dried over sodium sulfate. After vacuum removal ofsolvents, the product was purified by silica gel flash chromatographyusing a chloroform/methanol/acetic acid/water (85/15/1/1 v/v) system. Arepeat chromatography using a 0→5% methanol in chloroform (v/v) systemgave a more pure product. A total of 0.259 g of product (25% yield) wereisolated as a pair of regioisomers resulting from opening of theanhydride ring. Analysis on an NMR spectrometer was consistent with thedesired products.

Example 11 Preparation ofN-(3-Benzoylphenyl)-2-(carboxymethyl)hexadecanamide (Compound 16) andN-(3-Benzoylphenyl)-3-carboxyheptadecanamide (Compound 17)

The 3-aminobenzophenone, 0.500 g (2.53 mmol), was dissolved in 5.0 ml ofdry DMF along with 0.512 g (5.06 mmol) of TEA and 0.030 g (0.25 mmol) of4-dimethylaminopyridine. While stirring under argon, 0.826 g (2.79 mmol)of TDSA were added and the resulting solution was stirred at 45° C.overnight. The reaction was diluted with water and the desired productwas extracted with chloroform. After drying over sodium sulfate, thesolvent was removed and the product was purified on silica gel flashchromatography. The less polar impurities were eluted with chloroformand the product was eluted with a 2.5→5.0% methanol in chloroform (v/v)gradient. A total of 1.048 g of product were isolated with a partialresolution of the two regioisomers resulting from opening of theanhydride ring system. Analysis on an NMR spectrometer was consistentwith the desired products.

Example 12 Preparation ofN-(4-Benzoylphenyl)-2-(carboxymethyl)hexadecanamide (Compound 18) andN-(4-Benzoylphenyl)-3-carboxyheptadecanamide (Compound 19)

The 4-aminobenzophenone, 0.500 g (2.53 mmol), was dissolved in 7.0 ml ofdry DMF along with 0.512 g (5.06 mmol) of TEA and 0.030 g (0.25 mmol) of4-dimethylaminopyridine. While stirring under argon, 0.826 g (2.79 mmol)of TDSA were added and the resulting solution was stirred at 55° C. for80 hours. At this time, thin layer chromatography (TLC) revealed partialconversion to a less polar UV active product. The reaction was dilutedwith water and the desired product was extracted with chloroform. Afterdrying over sodium sulfate, the solvent was removed and the product waspurified on silica gel flash chromatography. The less polar impuritieswere eluted with chloroform and the product was eluted with a 2.5→5.0%methanol in chloroform (v/v) gradient. A total of 0.753 g of productwere isolated with a partial resolution of the two regioisomersresulting from opening of the anhydride ring system. Analysis on an NMRspectrometer was consistent with the desired products.

Example 13 Preparation of Monohexadecanamidopoly(ethylene glycol)₂₀₀Mono-4-benzoylbenzyl Ether (Compound 20)

Compound 5, 0.914 g (2.32 mmol), prepared according to the generalmethod described in Example 5, was dissolved in 10 ml of anhydrouschloroform with stirring under argon. TEA, 0.516 g (5.10 mmol), wasadded followed by the slow dropwise addition of 0.701 g (2.55 mmol) ofpalmitoyl chloride. The resulting mixture was stirred at roomtemperature overnight. The reaction was diluted with water and theproduct was extracted with chloroform. After drying over sodium sulfate,the solvent was removed under vacuum and the product was purified bysilica gel chromatography. A chloroform/methanol (95/5) solvent was usedto elute the product, yielding 382 mg of a viscous oil. Analysis on anNMR spectrometer was consistent with the desired product.

Example 14 Preparation of Mono-3-Carboxypropanamidopoly(ethyleneglycol)₂₀₀ Mono-4-benzoylbenzyl Ether (Compound 21)

Compound 5, 0.500 g (1.27 mmol), prepared according to the generalmethod described in Example 5, was dissolved in 5 ml of anhydrouschloroform along with 0.14 g (1.40 mmol) of succinic anhydride. Aftersolution was complete, 0.141 g (1.39 mmol) of TEA were added withstirring under argon. The resulting mixture was stirred at roomtemperature for 24 hours. The solvent was then removed under vacuum andthe product was purified on a silica gel flash chromatography columnusing a chloroform solvent, followed by a chloroform/methanol (95/5 to90/10 v/v) solvent gradient. Pooling of appropriate fractions andevaporation of solvent gave 447 mg of a viscous oil. Analysis on an NMRspectrometer was consistent with the desired product.

Example 15 Preparation of Hexadecyl 4-Benzoylbenzyl Ether (Compound 22)

1-Hexadecanol, 5.0 g (20.6 mmol), was dissolved in 10 ml of anhydrousTHF with warming, followed by slow addition of 0.840 g (21.0 mmol) of a60% dispersion of NaH in mineral oil. Once the hydrogen evolution wascomplete, 6.35 g (23.1 mmol) of BMBP, prepared according to the generalmethod described in Example 2, were added. The reaction mixture wasstirred at 50° C. under argon for one hour and then at room temperaturefor 16 hours. After this time the reaction was quenched with water andthe product was extracted with chloroform. After drying over sodiumsulfate, the solvent was removed under vacuum and the residue waspurified by silica gel flash chromatography using a hexane/ether (90/10)solvent. Appropriate fractions were pooled and evaporated to give 8.01 gof a waxy solid, an 88.9% yield. Analysis on an NMR spectrometer wasconsistent with the desired product.

Example 16 Preparation of Poly(ethylene glycol)₂₀₀ MonohexadecylMono-4-benzoylbenzyl Ether (Compound 23)

Compound 3, 1.00 g (2.54 mmol), prepared according to the general methoddescribed in Example 3, was dissolved in 10 ml of anhydrous THF under anargon atmosphere. Sodium hydride, 0.112 g (2.80 mmol) of a 60%dispersion in mineral oil, was added in portions while stirring on anice bath. The mixture was allowed to stir 20 minutes at roomtemperature, followed by the addition of 0.776 g (2.54 mmol) of1-bromohexadecane. The mixture was stirred overnight at roomtemperature. The reaction was quenched with water and the product wasextracted with chloroform. After drying over sodium sulfate and removalof solvent, the product was purified by silica gel flash chromatographyusing a chloroform/methanol/acetic acid/water (85/15/1/1 v/v) solvent aseluent. The appropriate fractions were pooled to give 1.357 g ofproduct, an 86% yield. Analysis on an NMR spectrometer was consistentwith the desired product.

Example 17 Preparation of Poly(ethylene glycol)₂₀₀Mono-15-carboxypentadecyl Mono-4-benzoylbenzyl Ether (Compound 24)

10-Hydroxyhexadecanoic acid, 0.785 g (2.88 mmol), was dissolved in 20 mlof anhydrous DMF in a dry flask under argon. Sodium hydride, 0.260 g(6.5 mmol) of a 60% dispersion in mineral oil, was then added and theresulting slurry was stirred at 60° C. for four hours. After this time,Compound 4, 1.24 g (2.62 mmol), prepared according to the general methoddescribed in Example 4, was added as a solution in 7 ml of DMF. Theresulting slurry was stirred at room temperature for 72 hours. Afterthis time, the reaction was quenched with water and the product wasextracted with chloroform. After drying over sodium sulfate, the productwas purified on a silica gel flash chromatography column. The column waseluted with chloroform/methanol (95/5 v/v) until the less polarimpurities were removed, followed by elution of the product withchloroform/methanol/acetic acid/water (90/10/1/1 v/v). The appropriatefractions were pooled and evaporated to yield 1.24 g of product, a 74%yield. Analysis on an NMR spectrometer was consistent with the desiredproduct.

Example 18 Preparation of Mono-15-carboxypentadecanamidopoly(ethyleneglycol)₂₀₀ Mono-4-benzoylbenzyl Ether (Compound 25)

Hexadecanedioic acid, 0.500 g (1.75 mmol), was dissolved in 5 ml ofanhydrous DMF with stirring under an argon atmosphere.N-Hydroxysuccinimide, 0.442 g (3.84 mmol) and dicyclohexylcarbodiimide,1.44 g (6.98 mmol), were added and the mixture was stirred for six hoursat room temperature. The resulting solid was removed by filtration andthe filter cake was washed with 1 ml of DMF. The solution was thenreacted with 0.747 g (1.90 mmol) of Compound 5, prepared according tothe general method described in Example 5, dissolved in 5 ml of DMF and0.389 g (3.84 mmol) of TEA. After stirring two hours at roomtemperature, TLC showed complete consumption of the starting amine. Theproduct was purified on a silica gel flash chromatography column byeluting less polar impurities using chloroform and elution of thedesired product using a chloroform/methanol/acetic acid/water (85/15/1/1v/v) solvent. The appropriate fractions were pooled and evaporated togive 1.356 g of product. Analysis on an NMR spectrometer was consistentwith the desired product.

Example 19 Preparation ofN-[3-Methacrylamido)propyl]-2-(carboxymethyl)hexadecanamide (Compound26) and N-[3-Methacrylamido)propyl]-3-carboxyheptadecanamide (Compound27)

N-(3-Aminopropyl)methacrylamide hydrochloride (APMA-HCl), 6.064 (33.9mmol), was dissolved in anhydrous methylene chloride along with 10.24 g(101 mmol) of TEA. TDSA, 10.0 g (33.7 mmol), was immediately added andthe mixture was stirred 48 hours at room temperature with moistureprotection from a drying tube. After this time, the reaction wasacidified with 1 N HCl, extracted with chloroform, and dried over sodiumsulfate. The product was purified on a silica gel chromatography columnusing a chloroform/methanol/acetic acid/water (85/15/1/1 v/v) solvent.The appropriate fractions were pooled, 100 ppm of phenothiazine wereadded, and the solvent was removed under reduced pressure to give 16.0 gof product as a pair of regioisomers resulting from opening of theanhydride ring. Analysis on an NMR spectrometer was consistent with thedesired products.

Example 20 Preparation of N-[3-(4-Benzoylbenzamido)propyl]methacrylamide(BBA-APMA) (Compound 28)

APMA-HCl, 120.0 g (0.672 mol), was suspended in 800 ml of chloroformalong with 25 mg of phenothiazine. The solution was cooled to below 10°C., followed by the addition of 172.5 g (0.705 mol) of BBA-Cl, preparedaccording to the general method described in Example 1. A solution of150.3 g (1.49 moles) of TEA in 50 ml of chloroform was prepared and thesolution was added dropwise to the above suspension over a 1-1.5 hourtime period while stirring on an ice bath. After completion of theaddition, the ice bath was removed and the solution was stirred for 2.5hours to complete the reaction. The mixture was then washed with 600 mlof 0.3 N HCl followed by 2×300 ml of 0.07 N HCl. The chloroform solutionwas then dried over sodium sulfate and the product was recrystallizedtwice using a toluene/chloroform (4/1 v/v) mixture. Phenothiazine, 25mg, was added prior to the second recrystallization to prevent prematurepolymerization. The yield was 212 g (90% yield) with a melting point of147-151° C. Analysis on an NMR spectrometer was consistent with thedesired product.

Example 21 Preparation ofN-(2-Mercaptoethyl)-3,5-bis(4-benzoylbenzyloxy)benzamide (Compound 29)

A photoactivatable chain transfer reagent was prepared in the followingmanner, and used in the manner described in Examples 22 and 24.3,5-Dihydroxybenzoic acid, 46.2 g (0.30 moles), was weighed into a 250ml flask equipped with a Soxhlet extractor and condenser. Methanol, 48.6ml, and concentrated sulfuric acid, 0.8 ml, were added to the flask and48 g of 3A molecular sieves were placed in the Soxhlet extractor. Theextractor was diluted with methanol and the mixture was heated at refluxovernight. Gas chromatographic analysis on the resulting product showeda 98% conversion to the desired methyl ester. The solvent was removedunder reduced pressure to give approximately 59 g of crude product. Thisproduct was used in the following step without further purification. Asmall sample was purified for NMR analysis, resulting in a spectrumconsistent with the desired product.

The entire methyl ester product from above was placed in a 2 liter flaskwith overhead stirrer and condenser, followed by the addition of 173.25g (0.63 mol) of BMBP, prepared according to the general method describedin Example 2, 207 g (1.50 mol) of potassium carbonate, and 1200 ml ofacetone. The resulting mixture was then refluxed overnight to givecomplete reaction as indicated by TLC. The solids were removed byfiltration and the acetone was evaporated under reduced pressure to give49 g of crude product. The solids were diluted with 1 liter of water andextracted with 3×1 liter of chloroform. The extracts were combined withthe acetone soluble fraction and dried over sodium sulfate, yielding 177g of crude product. The product was recrystallized from acetonitrile togive 150.2 g of a white solid, a 90% yield for the first two steps.Melting point of the product was 131.5° C. (DSC) and analysis on an NMRspectrometer was consistent with the desired product.

The methyl 3,5-bis(4-benzoylbenzyloxy)benzoate, 60.05 g (0.108 mol), wasplaced in a 2 liter flask, followed by the addition of 120 ml of water,480 ml of methanol, and 6.48 g (0.162 mol) of sodium hydroxide. Themixture was heated at reflux for three hours to complete hydrolysis ofthe ester. After cooling, the methanol was removed under reducedpressure and the sodium salt of the acid was dissolved in 2400 ml ofwarm water. The acid was precipitated using concentrated hydrochloricacid, filtered, washed with water, and dried in a vacuum oven to give58.2 g of a white solid (99% yield). Melting point on the product was188.3° C. (DSC) and analysis on an NMR spectrometer was consistent withthe desired product.

The 3,5-bis(4-benzoylbenzyloxy)benzoic acid, 20.0 g (36.86 mmol), wasadded to a 250 ml flask, followed by 36 ml of toluene, 5.4 ml (74.0mmol) of thionyl chloride, and 28 μl of DMF. The mixture was refluxedfor four hours to form the acid chloride. After cooling, the solvent andexcess thionyl chloride were removed under reduced pressure. Residualthionyl chloride was removed by four additional evaporations using 20 mlof chloroform each. The crude material was recrystallized from tolueneto give 18.45 g of product, an 89% yield. Melting point of product was126.9° C. (DSC) and analysis on an NMR spectrometer was consistent withthe desired product.

The 2-aminoethanethiol hydrochloride, 4.19 g (36.7 mmol), was added to a250 ml flask equipped with an overhead stirrer, followed by 15 ml ofchloroform and 10.64 ml (76.5 mmol) of TEA. After cooling the aminesolution on an ice bath, a solution of3,5-bis(4-benzoylbenzyloxy)benzoyl chloride, 18.4 g (32.8 mmol), in 50ml of chloroform was added dropwise over a 50 minute period. Cooling onice was continued 30 minutes, followed by warming to room temperaturefor two hours. The product was diluted with 150 ml of chloroform andwashed with 5×250 ml of 0.1 N hydrochloric acid. The product was driedover sodium sulfate and recrystallized twice from toluene/hexane (15/1v/v) to give 13.3 g of product, a 67% yield. Melting point on theproduct was 115.9° C. (DSC) and analysis on an NMR spectrometer wasconsistent with the desired product.

Example 22 Preparation of a Photoreactive Endpoint Copolymer ofAcrylamide and Fatty Acid Monomers (Compound 30)

Acrylamide, 0.640 g (9.00 mmol), was dissolved in 9 ml of THF, followedby the addition of 0.299 g (0.68 mmol) of Compounds 26 and 27, preparedaccording to the general method described in Example 19, 0.060 g (0.10mmol) of Compound 29, prepared according to the general method describedin Example 21, 9 μl (0.060 mmol) of N,N,N′,N′-tetramethylethylenediamine(TEMED), and 0.049 g (0.30 mmol) of 2,2′-azobisisobutyronitrile (AIBN).The solution was sparged two minutes with helium, two minutes withargon, and was then sealed and heated overnight at 55° C. The resultingsuspension was diluted with 5 ml of additional THF and added to diethylether, followed by filtration to isolate the solid. After drying in avacuum oven, 0.966 g of a white solid were isolated. Analysis of thepolymer revealed 0.073 mmol of BBA per gram of polymer.

Example 23 Preparation of a Photoreactive Random Copolymer of Acrylamideand Fatty Acid Monomers (Compound 31)

Acrylamide, 0.657 g (9.24 mmol), was dissolved in 9 ml of THF, followedby the addition of 0.307 g (0.70 mmol) of Compounds 26 and 27, preparedaccording to the general method described in Example 19, 0.036 g (0.10mmol) of Compound 28, prepared according to the general method describedin Example 20, 9 μl (0.060 mmol) of TEMED, and 0.026 g (0.16 mmol) ofAIBN. The solution was sparged two minutes with helium, two minutes withargon, and was then sealed and heated overnight at 55° C. The resultingsuspension was diluted with 5 ml of additional THF and added to diethylether, followed by filtration to isolate the solid. After drying in avacuum oven, 0.997 g of a white solid were isolated. Analysis of thepolymer revealed 0.086 mmol of BBA per gram of polymer.

Example 24 Preparation of a Photoreactive Endpoint Copolymer ofN-Vinylpyrrolidone and Fatty Acid Monomers (Compounds 32A-C)

N-Vinylpyrrolidone, 0.915 g (8.23 mmol), was dissolved in 3 ml of THF,followed by the addition of 0.271 g (0.618 mmol) of Compounds 26 and 27,prepared according to the general method described in Example 19, 0.070g (0.116 mmol) of Compound 29, prepared according to the general methoddescribed in Example 21, 1 μl (0.01 mmol) of TEMED, and 0.057 g (0.347mmol) of AIBN. This composition was designed to make TDSA 7 mole % ofthe monomers in the reaction mixture. The solution was sparged twominutes with helium, two minutes with argon, and was then sealed andheated overnight at 55° C. The polymer was precipitated by the additionof diethyl ether, followed by isolation with filtration. After drying ina vacuum oven, 1.10 g of a white solid were isolated. Analysis ofCompound 32A revealed 0.109 mmol of BBA per gram of polymer.

The above procedure was followed using the following quantities ofreagents in 4 ml of THF: N-vinylpyrrolidone, 0.433 g (3.90 mmol);Compounds 26 and 27, 0.507 g (1.16 mmol) Compound 29, 0.060 g (0.10mmol); TEMED, 3 μl (0.02 mmol); and AIBN, 0.049 g (0.298 mmol). Thiscomposition was designed to make TDSA 23 mole % of the monomers in thereaction mixture. After isolation following the above procedure, 0.808 gof a white solid were isolated. Analysis of Compound 32B revealed 0.083mmol of BBA per gram of polymer.

The above procedure was followed using the following quantities ofreagents in 3 ml of THF: N-vinylpyrrolidone, 0.181 g (1.63 mmol);Compounds 26 and 27, 0.759 g (1.73 mmol); Compound 29, 0.060 g (0.10mmol); TEMED, 1 μl (0.01 mmol); and AIBN, 0.049 g (0.298 mmol). Thiscomposition was designed to make TDSA 50 mole % of the monomers in thereaction mixture. After isolation following the above procedure, 0.705 gof a white solid were isolated. Analysis of Compound 32C revealed 0.102mmol of BBA per gram of polymer.

Example 25 Preparation of a Photoreactive Random Copolymer ofN-Vinylpyrrolidone and Fatty Acid Monomers (Compounds 33A-D)

N-Vinylpyrrolidone, 0.749 g (6.74 mmol), was dissolved in 8.8 ml of THF,followed by the addition of 0.224 g (0.511 mmol) of Compounds 26 and 27,prepared according to the general method described in Example 19, 0.027g (0.077 mmol) of Compound 28, prepared according to the general methoddescribed in Example 20, 1 μl (0.01 mmol) of TEMED, and 0.019 g (0.116mmol) AIBN. This composition was designed to make TDSA 7 mole % of themonomers in the reaction mixture. The solution was sparged two minuteswith helium, two minutes with argon, and was then sealed and heatedovernight at 55° C. The polymer was precipitated by the addition ofdiethyl ether, followed by isolation with filtration. After drying in avacuum oven, 0.353 g of a white solid were isolated. Analysis of theCompound 33A revealed 0.112 mmol of BBA per gram of polymer.

The above procedure was followed using the following quantities ofreagents in 3 ml of THF: N-vinylpyrrolidone, 0.362 g (3.26 mmol);Compounds 26 and 27, 0.621 g (1.42 mmol); Compound 28, 0.017 g (0.049mmol); TEMED, 1 μl (0.01 mmol); and AIBN, 0.012 g (0.073 mmol). Thiscomposition was designed to make TDSA 30 mole % of the monomers in thereaction mixture. After isolation following the above procedure, 0.770 gof a white solid were isolated. Analysis of Compound 33B revealed 0.052mmol of BBA per gram of polymer.

The above procedure was followed using the following quantities ofreagents in 3 ml of THF: N-vinylpyrrolidone, 0.196 g (1.76 mmol);Compounds 26 and 27, 0.791 g (1.80 mmol); Compound 28, 0.013 g (0.037mmol); TEMED, 1 μl (0.01 mmol); and AIBN, 0.009 g (0.055 mmol). Thiscomposition was designed to make TDSA 50 mole % of the monomers in thereaction mixture. After isolation following the above procedure, 0.708 gof a white solid were isolated. Analysis of Compound 33C revealed 0.048mmol of BBA per gram of polymer.

The above procedure was followed using the following quantities ofreagents in 7 ml of THF: N-vinylpyrrolidone, 0.188 g (1.69 mmol);Compounds 26 and 27, 1.792 g (4.09 mmol); Compound 28, 0.020 g (0.057mmol); TEMED, 1 μl (0.01 mmol); and AIBN, 0.014 g (0.085 mmol). Thiscomposition was designed to make TDSA 70 mole % of the monomers in thereaction mixture. After isolation following the above procedure, 0.879 gof a white solid were isolated. Analysis of Compound 33D revealed 0.058mmol of BBA per gram of polymer.

Example 26 Preparation of a Photoreactive Siloxane Copolymer ContainingFatty Acid Ligands (Compound 34)

An aminopropylmethylsiloxane-dimethylsiloxane copolymer, 5.00 g of a 6-7mole % amine monomer content, was dissolved in 50 ml of dry methylenechloride, followed by the addition of 0.79 g (7.81 mmol) of TEA. BBA-Cl,0.19 g (0.78 mmol), prepared according to the general method describedin Example 1, was then added and the mixture was stirred 3 hours at roomtemperature. TDSA, 0.924 g (3.12 mmol), was then added and the solutionwas stirred 24 hours at room temperature. The reaction was then dilutedwith water and the pH was adjusted to approximately 6 using 0.1 N HCl.The organic layer was removed and dried over sodium sulfate. The solventwas removed under reduced pressure and the resulting oil was dilutedwith hexane. The precipitate was removed by filtration and evaporationof the solvent gave 4.75 g of a viscous oil. Analysis of the polymerrevealed 0.013 mmol of BBA per gram of polymer.

Example 27 Fatty Acid Immobilization on an Amine Derivatized Surface

A polymer surface is derivatized by plasma treatment using a 3/1 mixtureof methane and ammonia gases (v/v). (See, e.g., the general methoddescribed in U.S. Pat. No. 5,643,580). A mixture of methane (490 SCCM)and ammonia (161 SCCM) are introduced into the plasma chamber along withthe polymer part to be coated. The gases are maintained at a pressure of0.2-0.3 torr and a 300-500 watt glow discharge is established within thechamber. The sample is treated for a total of 3-5 minutes under theseconditions. Formation of an amine derivatized surface is verified bysurface analysis using Electron Spectroscopy for Chemical Analysis(ESCA) and Time of Flight Secondary Ion Mass Spectrometry (TOF-SIMS).

TDSA is dissolved at a concentration of 30 mg/ml in a solvent compatiblewith both the polymer substrate and the anhydride. TEA, 1.5 equivalentsrelative to the anhydride, are added to the solution and the finalmixture is allowed to incubate with the amine derivatized surface for 24hours at room temperature to permit maximal coupling of the fatty acidto the surface. The final surface is then washed with fresh solvent toremove all unreacted materials and the final wash is a dilute acid washto remove any remaining TEA.

Example 28 Surface Modification of Selected Substrates with Reagents

Three polymers commonly-used as biomaterials were surface-modified withnovel compounds described above. The polymer substrates includedpolyethylene (PE), polyvinylchloride (PVC), and polyurethane (PU). Thesepolymers were obtained as flat sheets and used as 1×1 cm squares, 1 cmcircular disks or obtained in cylindrical form (tubes or rods) and usedas short segments. The shape and size of the part was chosen based onthe particular assay to be conducted with the coated substrates.

Coating solutions were prepared by dissolving the reagents atconcentrations ranging from 1-15 mg/ml in neat isopropanol (IPA) ordeionized water/IPA solutions. The reagents were applied to the polymersubstrates using dip coating methods. Parts were suspended vertically,immersed in the solution at 2 cm/sec, allowed to dwell for five seconds,and then withdrawn at a rate of 0.1 cm/sec. After removal of thesubstrate from the coating solution, it was air dried until the solventwas no longer visible, often within about 1 minute. The substrate withthe coating was then suspended midway between two opposed Dymax UVcuring lamps, each outfitted with a Heraeus Q402Z4 bulb. At the distanceof placement of the lamps, the parts received approximately 1.5 mW/cm²in the wavelength range 330-340 nm. The substrate was rotated at 3 rpmduring the two minutes of illumination to ensure that the surface wasevenly bathed in light. After illumination, the parts were removed fromthe lamp chamber and washed in IPA, using two sequential 30-minutewashes in fresh solvent. The coated samples were then stored in the darkat ambient temperature until used.

Example 29 Surface Analysis of Polymer Substrates Modified withCompounds 8, 9, 18, 19, 32 and 33

Three different techniques (staining, ESCA, and TOF-SIMS) were used toevaluate the surfaces of modified substrates to confirm the presence anduniformity of the compounds.

PE and PVC flat materials were modified with heterobifunctional reagents(Compounds 8, 9, 18, 19) and polymeric reagents (Compounds 32 and 33,having varying monomer compositions). Reagents were prepared in IPA at1.0 mg/ml and applied using the methods described in Example 28.

First, the coated materials were stained with Toluidine Blue 0, apositively-charged, visible-wavelength dye. Samples were immersed in asolution of the dye (0.02% w/v in water) for 30 seconds, removed fromsolution, and rinsed with DI water. This staining protocol was usefulfor identifying qualitatively the presence of each of the reagents onthe material surface. The results of the dye binding suggested that thesurface modification procedures were successful in immobilizing thereagents on the substrate surfaces. There was some variability in thedarkness of the stain, both from different reagents on the same materialand for the same reagents on different materials. The staining wasgrossly uniform to the naked eye over the surfaces of the material,suggesting that the reagent was not pooling or segregating when appliedto the surface and that the coverage of the surface was relativelyuniform.

ESCA was used to analyze quantitatively the surface chemical compositionof the modified substrates. PE and PVC modified with heterobifunctionalreagents (Compounds 8, 9, 18, 19) and polymeric reagents (Compounds 32and 33, having varying molar compositions) were analyzed with a PerkinElmer Model 5400 ESCA system using monochromatic Al X-rays with analysisat a 65 degree takeoff angle. Survey spectra were collected to calculatethe atomic concentrations in the surface.

The results of the ESCA measurements (Tables 1 and 2) on the surfacemodified materials were useful for indicating the presence and chemicalcomposition of the coatings. For the PVC substrate, the atomicconcentration of the chlorine atom (Cl) was used to determine whetherthe coating masked the substrate material. By comparing the amounts ofCl detected on the surface of the PVC after modification, it was clearthat the Cl was greatly reduced on the surface-modified substrates.Together with the results of the dye binding described above, thissuggested that the reagents covered the surface completely, but werethin enough to detect the underlying substrate. For the PE substrate,which in the uncoated state should have an atomic concentration of 100%carbon (as ESCA cannot detect H atoms), the modified and unmodifiedsamples could simply be compared using the carbon concentration. On allof the modified samples the carbon concentration was reduced by about20%. It was also evident that nitrogen was present on the surfaces ofthe modified PE and PVC, but not on the uncoated surfaces. This wasindicative of the nitrogen in each of the reagents. Finally, thesimilarity in the atomic concentrations of C, O, and N on the surfacesof PE and PVC samples modified with each compound supports the presenceand completeness of the coating. TABLE 1 Atomic Concentration summaryfor PE samples (atomic %). Sample [C] [O] [N] [Cl] [Si] [Na] Uncoated100 — — — — — Compound 33D 83.4 10.5 6.2 — — — Compound 33C 80.9 11.37.8 — — — Compound 33B 80.7 11.1 8.3 — — — Compound 32C 80.3 12.4 7.3 —— — Compound 32B 79.3 12.1 8.1 — 0.5 — Compounds 18, 19 83.8 13.9 2.3 —— — Compounds 8, 9 81.4 16.8 1.6 — — 0.2

TABLE 2 Atomic Concentration Summary for PVC samples (atomic %). Sample[C] [O] [N] [Cl] [Si] [Na] Uncoated 74.2 7.5 — 17.6 — 0.5 Compound 33D80.4 11.5 6.2 1.9 — — Compound 33C 78.8 12.1 8.8 0.4 — — Compound 33B77.8 11.6 9.1 1.3 — 0.1 Compound 32C 79.9 12.4 6.8 0.8 — — Compound 32B77.6 12.2 9.1 0.4 0.7 — Compounds 18, 19 83.4 12.8 2.4 1.4 — — Compounds8, 9 79.7 17.4 1.4 1.1 — 0.3

TOF-SIMS was conducted to ensure that the coatings were located on theoutermost surface of the substrates. TOF-SIMS is sensitive to thechemical structure within the outer 10 Å of a surface. TOF-SIMS wasperformed by Physical Electronics (Eden Prairie, Minn.) using a PhysicalElectronics model number 7200 instrument. Positive- and negative-ionspectra were recorded for each of the surfaces. In addition, scans ofthe surface were used to determine the uniformity of chemical fragmentswhich were indicative of the coatings (independent of the substratechemistry). The surfaces (substrates and coatings) analyzed by TOF-SIMSwere the same as those analyzed by ESCA, described above. For the coatedsubstrates, the TOF-SIMS spectra were substantially different from thespectra for the uncoated PE or PVC material. For example, there weremany chemical fragments containing nitrogen, which is not present ineither of the base materials. There were many high molecular weightfragments in the positive ion spectra (between 200 and 600 mass/chargeunits) associated with the heterobifunctional reagents (Compounds 8, 9and 18, 19). The polymer-based reagents (Compounds 32, 33) had regularrepeating fragment fingerprints indicative of the polymer backbone. Alsoconfirming that the reagents were present on the surfaces of thematerials, was that the fragment patterns for each compound were similaron the two different substrates. In addition, the scans of the surfaceto detect the presence of peaks uniquely associated with the coatingreagents indicated that the reagents were relatively uniformlydistributed over the surface of the substrate, further confirming theresults of the Toluidine Blue O staining tests described previously.

Example 30 Human Serum Albumin (HSA) Adsorption from Buffer and PlateletPoor Plasma

Adsorption of human serum albumin (HSA) from single protein buffersolution and from diluted human platelet poor plasma (PPP) onto thepolymer materials was quantified using radiolabeled protein. Fattyacid-free HSA (Sigma Chemical, St. Louis Mo.) was radiolabeled with ³Husing sodium borohydride techniques (Means and Feeney, Biochemistry 7,2192 (1968)). Buffer solutions of HSA were prepared by dissolvingunlabeled HSA to a concentration of 0.1 mg/ml in Tris-saline (TN) buffersolution (50 mM Tris, 150 mM NaCl, pH 7.5). The resulting solution wasthen spiked with an aliquot of the ³H-HSA such that the specificactivity was approximately 1000 dpm/μg HSA for the total solution.Plasma solutions were prepared using a commercially-available PPP(George King Biomedical; Overland Park, Kans.) prepared from bloodanticoagulated with sodium citrate (3.8%). Just prior to an adsorptionexperiment, the PPP was diluted 4:1 with phosphate buffered saline (10mM phosphate, 150 mM NaCl, pH 7.4; PBS) and then spiked with theradiolabeled HSA such that the specific activity was approximately 6000dpm/μg of HSA in the diluted plasma.

Adsorption experiments were conducted identically for both the bufferand PPP solutions containing ³H-HSA. Circular disks (1 cm) of thesurface-modified PE and PVC were placed in 20 ml scintillation vials;uncoated disks of the same materials were used as controls. The pieceswere hydrated in 2 ml of TN overnight at room temperature. On the day ofthe experiment, ³H-HSA solutions (buffer or PPP) were prepared asdescribed above. The hydration buffer was aspirated from the polymersamples and 1.0 ml of the radiolabeled HSA solution was added to thevial. The vials were gently agitated on an orbital shaker for 2 hours atroom temperature. The HSA solution was aspirated and 4 ml of TNTsolution (50 mM Tris, 150 mM NaCl, 0.05% Tween20, pH 7.5) were added toeach vial; the vials were shaken for 15 minutes at room temperature. TheTNT wash step was repeated two times and the disks were transferred toclean, dry scintillation vials. Two ml of THF were added to each vialand the samples were strongly agitated on an orbital shaker overnight.To each vial, 10 ml of Hionic Fluor were added and thoroughly mixed byvortexing. The vials were counted using a liquid scintillation counter(Packard 1900 CA). The surface concentration of HSA was calculated fromthese data using the specific activity of the HSA adsorption solutionand the surface area of the disks.

PE and PVC were modified with heterobifunctional and polymeric compoundsusing the same procedures as described in Example 28. The results of thebinding of ³H-HSA out of TN buffer solution onto the modified anduncoated PE and PVC materials are shown in Table 3. TABLE 3 Adsorptionof HSA from TN buffer onto modified PE and PVC surfaces Surfaceconcentration of HSA (μg/cm²) Surface PE PVC Uncoated 0.069 ± 0.0010.066 ± 0.000 Compound 32B 0.068 ± 0.001 0.051 ± 0.001 Compound 32C0.050 ± 0.001 0.036 ± 0.001 Compound 33B 0.071 ± 0.002 0.066 ± 0.001Compound 33C 0.054 ± 0.000 0.045 ± 0.006 Compound 33D 0.136 ± 0.0000.036 ± 0.005 Compounds 18, 19 0.128 ± 0.005 0.098 ± 0.006 Compounds 10,11 0.167 ± 0.006 0.168 ± 0.007 Compounds 14, 15 0.191 ± 0.001 0.200 ±0.010 Compound 8, 9 0.191 ± 0.010 0.159 ± 0.007

The results of HSA binding from buffer solution indicated that many ofthe polymeric reagents bound HSA at similar levels to uncoated surfaces,whereas the heterobifunctional compounds enhanced binding by 2- to3-fold over uncoated.

Example 31 HSA Binding from Plasma to PE Modified with Compounds 8, 9,18, 19, 30, 32, and 33

PE flat substrates were modified with Compounds 8, 9, 18, 19, 30, 32,and 33. Compounds 8, 9, 18, 19, 32, and 33 were prepared in IPA atconcentration of 1 mg/ml and Compound 30 was prepared in IPA/water(80/20 v/v), and substrates were coated following the procedure asdescribed in Example 28. HSA binding from PPP was measured as describedin Example 30; the specific activity was 2,003 dpm/μg. TABLE 4 HSAbinding from PPP onto PE Surface concentration Surface (PE) (μg/cm²)Uncoated 0.008 ± 0.001 Compound 32B 0.064 ± 0.012 Compound 33A 0.010 ±0.000 Compound 33B 0.168 ± 0.002 Compound 30 0.015 ± 0.000 Compounds 18,19 0.017 ± 0.002 Compounds 8, 9 0.012 ± 0.001

Example 32 HSA Binding from Plasma to PVC Modified with Compounds 8, 9,32, and 33

PVC flat substrates were modified with Compounds 8, 9, 32, and 33. Thecompounds were prepared in IPA at concentration of 1 mg/ml, and wereapplied to the substrates following the procedure as described inExample 28. HSA binding from PPP was measured as described in Example30; in this experiment the specific activity was 3,150 dpm/μg HSA. TABLE5 HSA binding from PPP onto PVC Surface concentration Surface (μg/cm²)Uncoated 0.0183 ± 0.0005 Compound 32C 0.0460 ± 0.0040 Compound 32B0.0420 ± 0.0010 Compound 33C 0.1720 ± 0.0120 Compound 33B 0.0830 ±0.0020 Compounds 8, 9 0.0296 ± 0.0010

Example 33 HSA Binding from Plasma to PE Modified with Compounds 14, 15

PE flat substrates were modified with Compounds 14, 15. The compoundswere prepared in IPA at concentrations ranging from 1-10 mg/ml andapplied as one coat or three coats, otherwise following the procedure asdescribed in Example 28. HSA binding from PPP was measured as describedin Example 30; specific activity of HSA was 5,636 dpm/μg in experiment#1 and #2. The results are shown in Table 6. TABLE 6 HSA binding fromPPP onto PE modified with Compounds 14, 15 Surface concentration of HSA(μg/cm²) Surface Experiment #1 Experiment #2 Uncoated PE 0.14 ± 0.0040.12 ± 0.005 1 mg/ml (3 coats) 0.16 ± 0.008 n.d.* 2.5 mg/ml (3 coats)0.28 ± 0.011 n.d. 5 mg/ml (1 coat) n.d. 0.48 ± 0.014 5 mg/ml (3 coats)0.48 ± 0.016 1.22 ± 0.046 7.5 mg/ml (1 coat) n.d. 0.67 ± 0.024 7.5 mg/ml(3 coats) 0.91 ± 0.018 1.02 ± 0.053 10 mg/ml (1 coat) n.d. 0.52 ± 0.01210 mg/ml (3 coats) 0.70 ± 0.037 1.18 ± 0.111*n.d. is not determined

The results of this experiment indicate that increasing theconcentration of applied reagent yields surfaces which show increasedbinding of HSA from PPP. In addition, increasing the number of coats ofreagent applied to the surface yields increased binding of HSA from PPP.

Example 34 HSA Binding from Plasma to PE Modified with Compounds 10, 11

PE flat substrates were modified with Compounds 10, 11. The compoundswere prepared in IPA at concentrations ranging from 1-15 mg/ml andapplied in three coats, otherwise following the procedure as describedin Example 28. HSA binding from PPP was measured as described in Example30; specific activity of the HSA in plasma was 5,977 dpm/1 g inExperiment #1 and 6,636 dpm/μg in Experiment #2. TABLE 7 HSA bindingfrom PPP onto PE modified with Compounds 10, 11 Surface concentration ofHSA (μg/cm²) Surface Experiment #1 Experiment #2 Uncoated PE 0.19 ±0.024 0.22 ± 0.017 1 mg/ml 0.43 ± 0.026 n.d. 2.5 mg/ml 0.25 ± 0.016 n.d.5 mg/ml 0.64 ± 0.030 n.d. 7.5 mg/ml 0.76 ± 0.084 n.d. 10 mg/ml 1.04 ±0.076 1.26 ± 0.092 12.5 mg/ml n.d. 0.91 ± 0.047 15 mg/ml n.d. 1.02 ±0.052

The results of this experiment indicate that increasing theconcentration of applied reagent yields increased HSA binding, althoughit appears as though the HSA binding reaches a plateau where furtherincreases in the reagent applied to the surface provide no additionalbenefit. This may indicate that the surface has become saturated withreagent.

Example 35 HSA Binding to PE Modified with Compounds 8, 9

PE flat substrates were modified with Compounds 8, 9. The compounds wereprepared in IPA at concentrations ranging from 1-10 mg/ml and applied asone coat or three coats, otherwise following the procedure as describedin Example 28. HSA binding from PPP was measured as described in Example30; specific activity in plasma was 6,045 dpm/μg HSA. TABLE 8 HSAbinding from PPP onto PE modified with Compounds 8, 9 Surfaceconcentration of HSA (μg/cm²) Surface One coat Three coats Uncoated PE0.196 ± 0.034 n.a. 1 mg/ml 0.194 ± 0.021 0.309 ± 0.039 2.5 mg/ml 0.3403± 0.034  0.642 ± 0.069 5 mg/ml 0.627 ± 0.067 0.692 ± 0.024 7.5 mg/ml1.043 ± 0.083 0.873 ± 0.063 10 mg/ml 1.071 ± 0.197 1.067 ± 0.013

These coatings on PE and PVC enhanced HSA binding from buffer and plasmaby as much as 10-fold. With some reagents (10, 11, 14, 15, and 8, 9),increasing concentration of coating solution produced surfaces withincreasing capacity to bind HSA. This plateau occurred near 7.5 mg/mlfor reagent 14, 15. For Compounds 8, 9, 10, 11, this plateau occurrednear 10 mg/ml.

Example 36 Fibrinogen (Fgn) Adsorption from PPP onto Modified Substrates

PE and PVC substrates were modified with Compounds 8, 9, 18, 19, 32, and33. The compounds were prepared in IPA at a concentration of 1.0 mg/mland applied as a single coat, otherwise following the procedure asdescribed in Example 28.

Adsorption of Fgn from human plasma (PPP) onto the control andsurface-modified materials was quantified by using ³H-Fgn. Fgn wasradiolabeled with ³H using sodium borohydride techniques (Means andFeeney, Biochemistry 7, 2192 (1968)) and stored frozen at −80° C. untilused. Plasma solutions of Fgn for adsorption experiments were preparedusing PPP (George King Biomedical; Overland Park, Kans.). On the day ofthe adsorption experiment, PPP was diluted 4:1 with TN buffer. Thediluted PPP was then spiked with an aliquot of the stock ³H-Fgn solutionto give a working solution with specific activity 1,816 dpm/μg Fgn.

Polymer samples (1 cm circular disks) were placed in 20 ml scintillationvials and hydrated overnight in 2.0 ml of TN at room temperature priorto protein adsorption. On the day of the experiment, the buffer solutionwas aspirated and 1.0 ml of the diluted PPP containing the ³H-Fgn wasadded to completely cover the polymer sample. The substrates wereincubated in the ³H-Fgn solution for 2 hours at 23° C. The PPP solutionwas aspirated and the substrates washed three times with TNT (15 minuteseach time). Disks were placed in clean scintillation vials, dissolvedwith THF, and counted for radioactivity as described in Example 30 forthe HSA adsorption experiments. Surface concentrations of Fgn werecalculated using the specific activity of the Fgn in the solution andthe surface area of the polymer samples. The experimental results of thefibrinogen absorption experiments are shown in Table 10. TABLE 10 Fgnadsorption to PE and PVC modified with Compounds 8, 9, 18, 19, 32, and33 Surface concentration of Fgn (μg/cm²) Surface treatment PE PVCUncoated 0.231 ± 0.152 0.269 ± 0.060 Compound 33C 0.148 ± 0.044 0.092 ±0.003 Compound 33B 0.160 ± 0.013 0.112 ± 0.013 Compound 32C 0.129 ±0.016 0.180 ± 0.005 Compound 32B 0.167 ± 0.016 0.249 ± 0.018 Compounds18, 19 0.131 ± 0.051 0.193 ± 0.029 Compounds 8, 9 0.194 ± 0.032 0.222 ±0.062

With these reagents, Fgn binding to modified surfaces was equal to orless than adsorption to uncoated surfaces. It is possible that theenhanced binding of HSA was responsible for reduced binding of Fgn.Surfaces that reduce the binding of Fgn are generally less likely toinduce subsequent unfavorable responses from blood, such as fibrinformation and platelet adhesion.

Example 37 Binding of Anti-HSA Antibodies to Modified PE Exposed to HSA

PE substrates were modified with Compounds 8, 9, 18, 19, 30, 31, 32, and33. The compounds were prepared in IPA at a concentration of 1.0 mg/mland applied as a single coat, otherwise following the procedure asdescribed in Example 28.

The binding of polyclonal anti-HSA antibodies was conducted using anELISA technique to determine whether bound albumin maintained nativestructure in the absorbed state. Sheep anti-(HSA) antibodies conjugatedto horseradish peroxidase (HRP) were obtained from Biodesign (Kennebunk,Me.). Polymer samples were hydrated with TN for 2 hours, and the proteinsolution was prepared with an HSA concentration of 1.0 mg/ml in TN. 1 mlof the protein solution was added to the samples and incubated for 2hours at room temperature. After the adsorption, the solution wasaspirated and the samples rinsed with TNT buffer. 1 ml of 1% BSA wasadded as a blocking step and incubated for one hour. The samples werewashed twice with TNT for 30 min. each. After the wash, the samples werebriefly rinsed with TN and incubated with the sheep-Ab-HRP in TN(diluted 1:2000), at room temperature for 1 hour with gentle agitation.The samples were washed 4 times with 3 mls TNT per vial by vortex. Thepieces were transferred to test tubes and 1 ml TMB/peroxide solution wasadded. The color was allowed to develop for 15 minutes. The absorbanceof the solutions was read at 655 nm using a spectrophotometer. Theabsorbance is directly proportional to the surface concentration of HRPand, therefore, also proportional to the surface concentration ofanti-HSA antibody bound to the substrate surfaces. TABLE 11 Results ofanti-albumin antibody binding to HSA exposed surface Surface Bound Ab(A₆₅₅) Uncoated 0.134 ± 0.005 Compound 32A 0.354 ± 0.030 Compound 32B0.335 ± 0.022 Compound 32C 0.338 ± 0.017 Compound 33A 0.311 ± 0.026Compound 33B 0.385 ± 0.034 Compound 33C 0.352 ± 0.020 Compound 30 0.332± 0.016 Compound 31 0.289 ± 0.025 Compounds 8, 9 0.456 ± 0.016 Compounds18, 19 0.488 ± 0.020

The results of anti-HSA antibody binding to HSA previously absorbed frombuffer to the uncoated and surface-modified materials indicated thatthere was little difference among the reagents tested. All surfacesbound high concentrations of antibody, about 3 to 4-fold higher thanuncoated surfaces.

Example 38 Platelet Attachment and Activation from Platelet Rich Plasma(PRP) on Modified PE and PVC

The surface-modified materials were incubated with platelet rich plasma(PRP) and then examined with a scanning electron microscope (SEM) todetermine the influence of surface chemistry on platelet attachment andactivation. Blood was collected fresh from human volunteers into 3.8%sodium citrate using 9:1 ratio of blood to anticoagulant. The blood wascentrifuged at 1200 rpm for 15 min. to separate PRP from blood. The PRPwas collected and kept at room temperature until used (less than 1hour). The test samples (1 inch squares) were placed in a 6-well plate,1 sample per well. To quantify the platelets in the plasma, a sample ofthe PRP was taken and diluted 1:100 with 1% ammonium oxalate. Acapillary tube was used to transfer a small amount of solution to ahemacytometer, and the sample was incubated in a covered petri dish for30 minutes for the platelets to settle. The platelets were counted undera phase contrast microscope and determined to be between 1.4-4.4×10¹⁴platelets/ml. The PRP solution was added onto the top of the samplesuntil the entire surface was covered, and the samples were incubated onehour at room temperature with no agitation. After incubation, the PRPwas removed carefully by aspiration and 3 mls of Tyrode's buffer (138 mMNaCl, 2.9 mM KCl, 12 mM sodium bicarbonate, pH 7.4) was gently added toeach well. The plates were agitated slightly on an orbital shaker for 15min.; the solution was changed and the wash repeated. The wash solutionwas aspirated and 2.0 ml of Karnovsky's fixative (25 mls formaldehyde+5mls 25% glutaraldehyde+20 mls of a solution of 23% NaH₂PO₄—H₂O+77%NaHPO₄ anhydrous) were added to each well. The plate was wrapped withparafilm and incubated overnight with slight agitation. The fixative wasaspirated and the samples were washed three times each with pure water,15 minutes for each wash. The samples were then dehydrated with anethanol series of 25, 50, 75, and 100%, for 15 minutes each. The sampleswere kept at 4° C. in 100% ethanol until mounted (up to 4 days). Sampleswere mounted and coated with Pd/Au and observed using a JEOL 840scanning electron microscope. Photos were taken of different areas onthe sample surface at several magnifications to give a representativeview of each sample. The platelets were counted and judged for degree ofactivation using morphological descriptions based on Goodman et alScanning Electron Microscopy/1984/I, 279-290 (1984).

The SEM results for two representative platelet attachment experimentsare shown in Tables 12 and 13. From the SEM photographs, surfacedensities of bound platelets were estimated. The lowest plateletdensities were found on the Compound 33C polymer consistently on bothsubstrates. The Compound 32C polymer also had low platelet densitiesconsistently. The predominant platelet morphologies are summarized inTable 13. Platelets that were rounded or dendritic were interpreted tobe less activated; whereas the platelets that were spreading or fullyspread and showed substantial aggregation were interpreted to be moreextensively activated. For PE, the uncoated substrate had the highestplatelet densities as well as the most fully spread platelet morphology.For PVC, the uncoated surface was poor but not the worst surface. TABLE12 Platelet Densities on modified surfaces (platelets/cm² × 10⁻⁶).Reagent PE PVC Uncoated 980 ± 50 650 ± 0  Compound 32C 420 ± 0  400 ± 30Compound 33C 220 ± 5  200 ± 30 Compounds 18, 19 900 ± 30 1000 ± 200Compounds 8, 9 400 ± 40 400 ± 0 

TABLE 13 Morphology of platelets attached to modified surfaces. SurfacePE PVC Compound Few aggregates, platelets N/A 32B mostly round ordendritic Compound Some aggregates, Few aggregates, platelets 32Cplatelets mostly round or mostly round or dendritic dendritic CompoundFew aggregates, platelets No aggregates, platelets 33C mostly round ordendritic mostly round Compound Few aggregates, platelets Someaggregates, platelets 33D mostly round or dendritic dendritic or spreaddendritic Compounds Many aggregates, most Many aggregates, most 18, 19platelets spread or fully platelets spreading or fully spread spreadCompounds Many aggregates, most Few aggregates, most 8, 9 plateletsspread or fully platelets are spreading spread Uncoated Many aggregates,most Few aggregates, platelets are platelets are fully spread spreaddendritic or fully spread

The polymeric reagents performed the best at reducing plateletattachment and activation on both substrates. The heterobifunctionalreagents 8 and 9 performed similarly to the polymeric reagent 32C. Theheterobifunctional reagents 18 and 19 were similar to or worse than theuncoated surface, depending on the substrate.

Example 39 Acute Dog Jugular Vein Implants with Catheters Modified withCompounds 6, 7

Surfaces modified with Compounds 6, 7 were tested using an acute, dog,jugular vein implant model. Surface-modified and control samples wereimplanted for one hour in the external jugular veins of 15-25 kgmixed-breed dogs. Attachment of ¹¹¹In-labeled, autologous platelets wasmonitored spatially and quantitatively in real time using gamma cameraimaging.

In each experiment, the dog was anesthetized with pentobarbital andsecured in a supine position. No anticoagulant was given to the animalsprior to or during the experiments. Ninety ml of blood was drawn intocitrate/dextrose (9:1 v/v) and the platelets were isolated and labeledwith ¹¹¹In-oxine. The labeled platelets were reinfused into the dog andallowed to circulate for 20 minutes. In quick succession, one rodmodified with a fatty acid derivative and one uncoated control rod wereimplanted bilaterally in the left and right external jugular veins. Byusing an uncoated control rod in each experiment, any variability in theresponse of individual animals to the implanted materials was accountedfor. Immediately after insertion of the rods, the neck region of the dogwas monitored continuously for one hour with a Picker 4/15 digital gammacamera to follow in real time the attachment of platelets onto the rods.The gamma camera allowed both digital quantification and spatialresolution of the radioactive counts. The data collected with the camerawas transferred to a dedicated microcomputer to calculate the relativeplatelet adhesion rates on the coated and control materials. After theone hour scan, the animal was heparinized systemically, to stop anyadditional thrombogenesis, and euthanized with an intravenous injectionof KCl. Each jugular vein was exposed and opened longitudinally toreveal the rod in place in the vein. After the rods were photographed,they were removed and the thrombus was stripped, lyophilized andweighed. TABLE 14 Comparison of platelet attachment on PU modified withCompounds 6, 7. Platelet attachment Surface rate versus control UncoatedPU 1.00 ± 0.43 Modified with Compounds 6, 7 0.39 ± 0.35

The coated PU surface performed significantly better than the uncoatedsurface, reducing platelet adhesion in this acute test of bloodcompatibility.

Example 40 Five-Month Sheep Mitral Valve Implants Using ModifiedSilicone Rubber Heart Valves

Silicone rubber (SR) heart valves are modified with reagents 14, 15. Thereagent is prepared at 5 mg/ml in IPA and applied, using procedures asdescribed in Example 28, in three coats to the surface of the SRportions of a polymeric, tri-leaflet valve. The valves are sterilizedusing ethylene oxide and implanted in the mitral position in juvenilesheep using procedures described previously Irwin, E. D., et al, J.Invest. Sur. 6, 133-141 (1993). Three valves treated with the reagentsare implanted. Valves are left in place for approximately 150 days. Atthe end of the implant period, the sheep are sacrificed and the heartsare explanted. The valve, including the surrounding heart tissue isremoved and placed in buffered formalin. The valves are examinedvisually and photographed.

The appearance of the explanted valve leaflets should be improved by thecoating. The coated valves should have minimal thrombus on the surfaceof the leaflets, whereas the uncoated SR valves should have substantialthrombus covering much of the surface of the leaflets. Furthermore, thethrombus present on the surface may be significantly mineralized, afurther detrimental outcome that would potentially shorten the usablelifetime of the valve.

1-56. (canceled)
 57. A passivating biomaterial comprising a surfaceprepared by (a) providing a surface derivatized with a nucleophilicspecies, (b) reacting the surface with a reactive molecule underconditions suitable to react the reactive molecule with the nucleophilicspecies in order to form a bifunctional aliphatic acid attached to thesurface by a covalent linkage.
 58. A biomaterial according to claim 57wherein the biomaterial is selected from the group consisting ofpolyolefins, polystyrenes, poly(methyl)methacrylates,polyacrylonitriles, poly(vinylacetates), poly (vinyl alcohols),chlorine-containing polymers such as poly(vinyl)chloride,polyoxymethylenes, polycarbonates, polyamides, polyimides,polyurethanes, phenolics, amino-epoxy resins, polyesters, silicones,cellulose-based plastics, and rubber-like plastics.
 59. A passivatedbiomaterial surface, comprising a biomaterial surface having covalentlyattached thereto a reagent according to claim 57, the surface beingpositioned in vivo under conditions suitable to permit albumin moleculesto be attracted and bound thereto in order to passivate the surface. 60.A medical article fabricated from a passivating biomaterial according toclaim
 59. 61. A medical article according to claim 60, wherein thearticle comprises a blood-contacting medical device for in vivoapplication.
 62. A passivated biomaterial surface comprising the surfaceof claim 59 having a proteinaceous material bound thereto.